Method for Making Mate-Pair Libraries

Abstract

The present disclosure provides methods for generating mate-pair libraries using a recombinase/recombination site system. The method allows for increased insert size, improved efficiency and simplicity of the steps involved, and improved data generation. Mate-pair libraries are helpful in providing positional information for the assembly of sequence data from short read sequencing platforms. The disclosure also embodies the mate-pair libraries as generated from these methods.

1 . A method for making a mate-pair library, the method comprising:
 a) fragmenting target DNA, thereby generating fragmented target DNA fragments;   b) size-selecting the fragmented target DNA, thereby generating size-selected DNA;   c) ligating a forward adaptor oligonucleotide and a reverse adaptor oligonucleotide to the size-selected DNA, wherein the forward adaptor oligonucleotide comprises a recombinase recombination site and a forward primer binding site and the reverse adaptor oligonucleotide comprises a recombinase recombination site and a reverse primer binding site, thereby generating adaptor-ligated size-selected DNA comprising ligated adaptor oligonucleotides at both ends of the fragments, wherein:
 the recombinase recombination site in the forward adaptor oligonucleotide, when ligated to the size-selected DNA, is distal to the size-selected DNA relative to the forward primer binding site; 
 the recombinase recombination site in the reverse adaptor oligonucleotide, when ligated to the size-selected DNA, is distal to the size-selected DNA relative to the reverse primer binding site; 
 the recombinase recombination sites are oriented in the adaptor-ligated size-selected DNA such that, when contacted with a recombinase, one of the recombinase recombination sites is excised and the adaptor-ligated size-selected DNA is circularized; and 
 the primer binding sites are oriented in the adaptor-ligated sized-selected DNA such that, after recombination, second fragmentation and recircularization, the forward and reverse primer binding sites are oriented toward each other so the amplification can occur; 
   d) removing the nick between DNA fragment and adapter;   e) contacting the adaptor-ligated size-selected DNA with the recombinase to form circularized DNA comprising the forward and reverse primer sites in opposing directions and separated by a recombinase recognition/recombination site;   f) removing non-circularized DNA from the circularized DNA or digesting the non-circularized DNA;   g) fragmenting the circularized DNA, thereby generating linear DNA fragments comprising the forward and reverse primer sites in opposing directions flanked by target DNA;   h) self-ligating the linear DNA fragments, thereby forming re-circularized DNA comprising the forward and reverse primer sites; and   i) amplifying the re-circularized DNA with the forward and reverse primer, thereby forming a mate-pair library.   
     
     
         2 . The method of  claim 1 , wherein the recombinase is a Cre recombinase and the recombinase recombination sites are lox sites. 
     
     
         3 . The method of  claim 1 , wherein the fragmenting (step g) comprises cutting the circularized DNA with a restriction enzyme. 
     
     
         4 . The method of  claim 3 , wherein the recognition sequence of the restriction enzyme is four contiguous base pairs. 
     
     
         5 . The method of  claim 1 , wherein the fragmenting (step g) comprises random shearing. 
     
     
         6 . The method of  claim 5 , wherein ends of the linear DNA fragments generated in step g are treated to generate blunt ends. 
     
     
         7 . The method of  claim 1 , wherein the amplifying (step i) comprises a polymerase chain reaction (PCR). 
     
     
         8 . The method of  claim 1 , further comprising sequencing the mate-pair library. 
     
     
         9 . The method of  claim 8 , further comprising aligning or assembling from data generated from sequencing. 
     
     
         10 . The method of  claim 9 , wherein the fragmenting (step g) comprises cutting the circularized DNA with a restriction enzyme, before aligning or assembling, determining the location of the recognition sequence of the restriction enzyme in sequencing reads and trimming the bases beyond the recognition site to avoid chimeric read. 
     
     
         11 . The method of  claim 1 , wherein, following the fragmenting (step a), and before the ligating (step c), ends of the fragmented target DNA are treated to generate blunt ends. 
     
     
         12 . The method of  claim 1 , wherein the target DNA is genomic DNA. 
     
     
         13 . The method of  claim 1 , wherein the fragmented target DNA is size-selected for DNA at any range up to 90 kb. 
     
     
         14 . A mate-pair library as generated in  claim 1 . 
     
     
         15 . A mate-pair library as generated in  claim 4 , characterized in that each member of the library comprises one or more recognition sequences of one or more restriction enzymes.